Project Summary In order to convey information, communication requires both signal and silence. In biology this is achieved through signaling enzymes that are dynamic and reversible, allowing itself to be both activated and inactivated. As a prototypical signaling kinase, Protein Kinase A, PKA, has been observed to be both activated and inactivated in a matter of seconds, which is essential for its roles in key physiological processes such as regulating heart contractility and the oscillatory release of insulin by the pancreas. PKA is inactive when its catalytic (C) and regulatory (R) subunits are bound in a closed confirmation and PKA is activated by the binding of the second messenger cyclic adenosine monophosphate, cAMP, to the regulatory subunit prompting the release of the catalytic subunit. While it had been assumed that equilibrium binding of cAMP would be commensurate with the activation and inactivation of PKA, both the dissociation of cAMP from the regulatory subunit and re-association of the regulatory and catalytic subunits are far too slow in vitro to agree with the fast inactivation observed in cells. The work proposed in this fellowship will examine the hypothesis that the inactivation of PKA requires the direct removal of cAMP by phosphodiesterases, PDEs, and the dephosphorylation of the regulatory subunit by the phosphatase Calcineurin, CN. This hypothesis will be tested with the following two specific aims: ? First, the kinetic effects of disrupting the ability of PDE to directly degrade cAMP bound to PKA will be measured at both the molecular and cellular levels. ? Second, in vitro and live-cell quantification of PKA activity will be used to evaluate the role of PKA RII dephosphorylation by CN in PKA inactivation. Furthermore, live-cell observation of PKA dependent changes in mitochondria morphology will allow us to characterize the downstream functional effects of RII dephosphorylation by CN. The results from these studies will answer a long standing question in the field of PKA signaling dynamics.